About 12.6 million units (including approximately 643,000 autologous donations) of whole blood are donated in the United States each year by approximately eight million volunteer blood donors. These units are transfused to about four million patients per year. Typically, each donated unit of blood, referred to as whole blood, may be separated into multiple components, such red blood cells, plasma, clotting factors, gamma globulin and platelets. The need for blood is great: on any given day, approximately 32,000 units of red blood cells are needed. Accident victims, people undergoing surgery and patients receiving treatment for leukemia, cancer or other diseases, such as sickle cell disease and thalassemia, all utilize blood.
Because patients seldom require all of the components of whole blood, it is the usual practice in blood banks to separate the blood into components and transfuse only that portion needed by the patient for a specific condition or disease. This treatment, referred to as xe2x80x9cblood component therapy,xe2x80x9d allows several patients to benefit from one unit of blood.
Whole blood is a living tissue that circulates through the heart, arteries, veins and capillaries, carrying nourishment, electrolytes, antibodies, heat and oxygen to the body tissues. Whole blood is comprised of red blood cells, white blood cells and platelets suspended in a proteinaceous fluid termed blood plasma. If blood is treated to prevent clotting and permitted to stand in a container, red blood cells will settle to the bottom of the container; the plasma will remain on top and the white blood cells will form a layer on top of the red blood cells. A centrifuge is commonly used to hasten this separation. The platelet-rich plasma is then removed and placed into a sterile bag for further processing to separate, for example platelets, clotting factors, albumin, immunoglobulin and the like.
Red blood cells contain hemoglobin, a complex iron-containing protein that carries oxygen throughout the body and gives blood its red color. The percentage of blood volume that is composed of red blood cells is called the xe2x80x9chematocrit.xe2x80x9d The average hematocrit is and adult male is 47%. There are about one billion red blood cells in two or three drops of blood, and, for every 600 red blood cells, there are about 40 platelets and one white blood cell. Manufactured in the bone marrow, red blood cells are continuously being produced and broken down and removed by the spleen after an average 120 days in the circulatory system. Red blood cells are prepared from whole blood by removing the plasma and can raise the patient""s hematocrit while minimizing and increase in blood volume, which is especially important to such patients as those with congestive heart failure. Patients benefiting most from transfusions of red blood cells include those with chronic anemia from disorders such as kidney failure, malignancies, gastrointestinal bleeding and acute blood loss as from trauma or surgery. Red blood cells may be treated and frozen for extended storage up to ten years.
Storage of these components varies. Improvements in cell preservation solutions over the last 15 years have increased the refrigerated shelf life of whole blood or red blood cells from 21 to 42 days. Plasma can be frozen and kept much longer. The isolated proteins such as clotting factors may be freeze dried for indefinite shelf life.
Platelets or thrombocytes are very small cellular components of blood that are programmed to aggregate in various conditions. Platelets are produced in the bone marrow and survive in the circulatory system for an average of nine or ten days before being removed from the body by the spleen. Platelets are vital to life, because they help prevent massive blood loss from trauma and the blood vessel leakage that occurs during normal daily activity.
Platelet transfusions are an integral part of the support of patients at risk of bleeding. Platelets used for transfusion can come from two sources: platelet concentrates derived from units of whole blood, termed random donor platelet concentrates, or apheresis platelets obtained from a single donor by plateletpheresis, a technique of continuous separation of platelets from a donor, with simultaneous reinfusion of blood minus platelets back into the donor. A unit of platelets is defined as the concentration of platelets separated from a single unit of whole blood and suspended in a small amount of plasma. The accepted unit contains no fewer than 5.5xc3x971010 platelets suspended in 40-70 ml of plasma. The recommended dosage of platelets is one unit per 10 kilograms body weight. This dosing schedule can be used for infants, children or adults to yield an expected increment in platelet count of 5,000-10,000 per microliter per unit of platelets transfused. The smaller the patient, the larger the relative dose. Thus an infant or small child may require an increase to 25,000 to 50,000 per unit of platelets.
Whatever the mode of collection or use, platelet storage poses problems that are not found with the storage of whole blood or other components. It is noted above that whole blood, red and white cells may be stored at 4xc2x0 C. for weeks. Platelets, which are programmed to aggregate and must be able to aggregate as part of their function, will aggregate in cold storage and when allowed to settle. Therefore, the standard means of storage of platelets is at room temperature, approximately 20 to 24xc2x0 C., with gentle agitation. Even under these conditions, platelets lose function by five days.
An additional problem is bacterial contamination. While blood is drawn under the most stringent aseptic techniques, invariably a tiny number of bacteria may enter the collection bag. Additionally, white blood cells may have scavenged bacteria. If these cells should rupture, bacteria may be released. Reported organisms include Staphylococcus epidermidis, Staphylococcus aureus, bacillus sp., micrococcus sp., streptococcus sp., klebsiella sp. and Salmonella sp. The gold standard for detection of contamination is a negative Petri culture at two weeks. A reported prevalence of contamination of whole blood for transfusion is 48.5 per 100,000. The situation for platelets is worse: platelet bacterial contamination is ten times greater than for blood. It is thought that this higher prevalence is due to the fact that platelets are stored at room temperature, which favors bacterial growth. The United States FDA reports 37 deaths since 1996 due to contaminated platelets, while the incidence in France is about four deaths per year.
A need remains to provide compositions that will increase the survival time of platelets and reduce bacterial contamination.
Platelets stored under previously known conditions lose function by undergoing spontaneous activation so that by five days only about 5% of the platelets are functional. This invention provides pentose to be added to stored platelets in concentrations ranging from 50 nM to 15 xcexcM, more preferably from 100 nM to 5 xcexcM. Such treated platelets retain normal function for as long as five days and show significant function at ten days of storage. An unexpected and additional advantage of pentose addition is the inhibition of bacterial growth.
Platelet function is measured by (1) internal protein expression on the cell membrane in response to challenge with an activation-inducing agonist; (2) ability to aggregate when challenged by an agonist; and (3) adenosine triphosphate secretion. Internal protein expression may be measured by conjugation of a molecule with a fluorescent dye, followed by sorting in a fluorescent cell sorter. In general, it is preferable to use two monoclonal antibodies, one that binds a cell surface molecule expressed and a second that binds a cell surface molecule that is expressed only after activation. Each monoclonal antibody is conjugated to a different colored dye, that can be distinguished by spectrofluorometry. In the preferred embodiment, the normally expressed cell surface molecule is GPIIbIIIa; the cell surface molecule expressed after activation is P-selectin. It is well know in the art to make monoclonal antibodies to proteins. U.S. Pat. No. 5,470,738, issued in 1995 to Freilich et al, is one example of a method of making monoclonal antibodies to GPIIIa. Another anti-platelet monoclonal antibody is that to GP IV, as disclosed by U.S. Pat. No. 5,231,025, issued in 1993 to Gralnisch. However, it is most convenient to purchase antibodies commercially from such companies as Becton-Dickinson (Philadelphia).
A basal measurement of spontaneous activation is obtained and the cells are then challenged with an agonist. The difference between cells challenged to activate minus the basal measurement is a measure of cell function. In the preferred embodiment, the agonist is 10 nM thrombin.
Another parameter of platelet function is the ability to aggregate when challenged by an agonist. The platelet suspension is dense and milky white. Aggregation and subsequent settling of the aggregates can be estimated visually, or measured with a densitometer.
Yet another measure of platelet function is the secretion of ATP. Platelets that are able to function well are able to secrete ATP while cells that have already been activated or have lost function in other ways cannot secrete ATP.
For purposes of describing this invention, the following terms have these meanings:
Agonist means any ligand that will bind to a cell surface protein of a platelet and cause activation of platelet function. Agonists include thrombin, epinephrine, ADP and collagen.
Pentose means a five carbon sugar which is D-Ribose or xylulose or a five-carbon precursor of ribose. The pentose related alcohol xylitol is also included in the definition of pentose.
Standard platelet storage conditions means suspension at a concentration of about 10xc3x97105 to 10xc3x971010 platelets per milliliter in a balanced, isotonic salt solution with an initial pH of at least 7, agitated or rotated gently, in a gas permeable storage bag, kept at room temperature.